Asymmetric PCR coupled with post-PCR characterization for the identification of nucleic acids

ABSTRACT

The present invention provides methods, compositions, and kits for quantification and identification of target nucleic acid sequences, either in pure solutions or from mixtures of various nucleic acids. In other aspects, the invention provides compositions and methods for HCV genotyping.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of the following UnitedStates Provisional Patent Applications:

-   -   Application Ser. No. 60/695,991, filed Jun. 30, 2005;    -   Application Ser. No. 60/696,253, filed Jun. 30, 2005;    -   Application Ser. No. 60/696,293, filed Jun. 30, 2005; and    -   Application Ser. No. 60/696,303, filed Jun. 30, 2005.        Each of these specifications are hereby incorporated by        reference in their entirety.

FIELD OF THE INVENTION

The current invention relates to the fields of nucleic acid chemistryand nucleic acid identification. More specifically, the inventionrelates to methods and compositions for amplifying and classifyingspecific nucleic acid sequences, which can be used for such purposes asdiagnostics.

BACKGROUND OF THE INVENTION

Numerous examples of the need for quick and reliable nucleic acidclassification/identification exist, especially in fields such asmedicine. For example, many diseases and infections are caused by anumber of, often related, pathogens. While the disease symptoms maypresent as similar, it can be of utmost importance to determine theactual causative pathogen in order to present an effective treatment.Not only is this true in terms of differentiation between differentinfectious species, but is also true, and can be even more difficult toresolve, when trying to discriminate between closely related agents,e.g., different strains of a pathogen such as the subtypes of hepatitisC virus (HCV).

Additionally, quick and reliable means of genotyping can be helpful indetermining allele composition within and amongst individuals. Forexample, reliable classification of particular alleles in an individualcan help in genetic counseling in humans and can even help in planningprophylactic treatment in instances when specific alleles are detected.Identification of particular alleles is also extremely useful inperforming marker assisted selection, e.g., crop or animal breedingprograms, identifying or genotyping pathogens and other organisms.

A number of different methods currently exist for detecting,identifying, genotyping, or quantifying various nucleic acids. Many ofthese rely on techniques that involve various binding actions betweennucleic acid probes and the nucleic acid being examined such asrestriction length fragment polymorphism analysis, sequencing, cleavageof probes that only occurs when specific target sequences are present,and the like.

However, there is a constant need for faster, simpler, and more flexibleanalysis tools. Ideal methods for classifying or genotyping of nucleicacid sequences would be easy to use and would involve the fewestmanipulations of the components needed, thus, decreasing instances oferror and reducing costs. The current invention provides these and otherbenefits which will be apparent upon examination of the currentspecification, claims, and figures.

SUMMARY OF THE INVENTION

In various aspects herein, the invention comprises methods foridentifying one or more nucleic acid targets in a sample (e.g., a bloodsample or urine sample from a subject and/or a mixed sample ofbiological isolate(s) such as nucleic acid sample(s) in solution from asubject). Such methods comprise: performing asymmetric kinetic PCR in areaction mixture containing one or more labeled 5′-nuclease probes andone or more labeled hybridization probes (wherein such 5′-nucleaseprobes can be the same as the hybridization probes or wherein the probescan be different from one another, e.g., in sequence, in labeling,etc.); monitoring one or more growth curves from the kinetic PCR (kPCR),e.g., by tracking indicators such as fluorescence, to construct suchgrowth curves; modifying temperature of the reaction mixture after thekPCR (e.g., over a range of temperatures, either increasing ordecreasing and either over a continuous range or over a number ofdiscrete temperatures) to cause a change in association between thelabeled hybridization probes and the nucleic acid targets (e.g., meltingor annealing); monitoring one or more fluorescent signals (or, in someembodiments, signals such as radiation, etc.) generated from the labeledhybridization probes, thereby producing a melting curve or annealingcurve; and, correlating the melting curve or annealing curve, thusproduced, to standard melting or annealing curves of completely orpartially complementary probes of known nucleic acid targets or the sameprobe tested against a known sample, thus, identifying the nucleic acidtargets in the sample. The standard melting or annealing curves canoptionally be determined from actual performance of the curves under setconditions or can be predicted based upon the nucleic acid compositionsof the hybridization probes and the targets under set conditions withoutactual performance of the curve.

In some embodiments of such methods “identifying the one or more nucleicacid targets” can involve identifying organisms or organism strainshaving the target nucleic acid. For example, in various embodiments,identifying can entail identification of (or the presence of) particularbacteria or bacterial strains (e.g., staphylococci species, Mycobacteriaspecies, Borrelia species, various enterococcus species, various E. colistrains, and the like), particular viruses or viral strains (e.g., HCV,HIV, influenza, HPV, HBV), particular fungi or fungal strains, or thepresence of particular alleles or haplotypes (e.g., as used in geneticcounseling to detect the presence and/or type of particular alleles).

In the various embodiments in the asymmetric kinetic PCR herein, theratio of the first primer to the second primer can be selectivelymanipulated. For example, in some embodiments, the asymmetric kPCRcomprises a first primer and at least a second primer, wherein theamount of the first primer is greater than the amount of the secondprimer (i.e., the limiting primer). Also, some embodiments comprisewherein the one or more hybridization probe (which is complementary tothe strand produced by the first, or non-limiting, primer in theasymmetric kPCR) exists in the reaction mixture in a greater amount thanthe second, or limiting, primer. For example, in some embodiments, theratio (of first primer to second primer) can comprise at least 2:1, atleast 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, atleast 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, atleast 50:1, at least 100:1, or at least 200:1 or more, depending upon,e.g., the desired end ratio of target nucleic acid strands (e.g., ratioof one strand to the other upon amplification).

In some embodiments, the methods comprise performing an asymmetric kPCR,followed by a thermal melting/annealing step, in the presence of anamplification indicator (e.g., an indicator of kinetic PCR amplificationof the target sequences) that comprises a fluorescently labeled5′-nuclease probe or otherwise labeled 5′-nuclease probe. Such probesmay be at least substantially complementary to, and hybridize with, thetarget nucleic acid sequences. In various embodiments, amplification isindicated by an increase in fluorescence, while in yet otherembodiments, amplification is indicated by a decrease in fluorescence.

In certain embodiments, the same probe(s) are used as the 5′-nucleaseprobes and as the hybridization probes, thereby simplifying andstreamlining the method steps. Thus, for example, the 5′-nuclease probescan be the same probes as the hybridization probes used to generatethermal melting/annealing curves as well. However, in yet otherembodiments, the fluorescently labeled 5′-nuclease probes can bedifferent probe(s) than the hybridization probes, e.g., in sequence, inlabeling, etc.

In certain embodiments, the probes (e.g., the hybridization probesand/or the 5′-nuclease probes) can be completely complementary to aregion of a nucleic acid target. In other embodiments, the probes can bepartially complementary to a region of a nucleic acid target. Thus, forexample, if a sample contained a number of different bacterial speciesall of which would have a target region amplified, but which targetregion comprised a different sequence in each species, a hybridizationprobe (whether or not it is the same as the 5′-nuclease probe) can becompletely complementary to one of the species' regions and partially ornot at all complementary to any of the other species' sequences; or theprobe can be not completely complementary to any of the species'sequences while partially or not at all complementary to each of theother species' sequences, etc.

In certain embodiments of the invention, the change in associationbetween the hybridization probe and the nucleic acid target can cause achange (e.g., increase) in fluorescence (which can then be optionallydetected and quantified). In yet other embodiments, the change inassociation between a hybridization probe and a nucleic acid target cancause a decrease in fluorescence, which also can be detected andquantified.

The hybridization probes used in the methods herein can optionally bepresent in the reaction mixture during amplification of the targetnucleic acid (e.g., as when the hybridization probes are the same probesas the 5′-nuclease probes or as when the probes are different, but areboth present in the reaction mixture prior to amplification), or in someembodiments, the hybridization probes can be not present during theamplification of the target nucleic acid, e.g., as when thehybridization probes do not comprise the same probes as the 5′-nucleaseprobes and are added after kPCR.

The monitoring in the embodiments herein can occur over a range oftemperatures, e.g., over a continuous range, or at discrete temperaturepoints within a range.

In detecting and quantifying the kinetic PCR amplification of the targetnucleic acids via the 5′-nuclease probes, a change in at least a firstfluorescence can be monitored, while detecting and quantifying of thechange in association of the hybridization probe with the target nucleicacid can be monitored by change in different fluorescence(s). Suchdifferent fluorescence(s) can optionally arise from different probes. Inthe embodiments wherein there are different probes (i.e., wherein the5′-nuclease probe is different from the hybridization probe), each onecan optionally be measured by a different fluorescence (e.g., fromdifferent fluorescent dyes) or other indicator. Other embodiments caninvolve measuring the same fluorescence for the kinetic PCR growth curveand for the change in association in the hybridization/melt curve sincesuch curves produce the fluorescence at different times in the reactionsequence.

In some aspects, herein, the invention comprises methods wherein the oneor more nucleic acid targets comprise a hepatitis C virus (HCV) nucleicacid (e.g., a nucleic acid from any HCV, such as HCV 1a, 1b, 2a, 2b, 3a,3b, 4, 5, 6, or any other type, subtype and/or genotype). Thus, in suchembodiments, identifying the HCV nucleic acid target identifies an HCVstrain in the sample. Furthermore, in such embodiments, thehybridization probe(s) is substantially complementary with an HCV straingenotype (or with more than one HCV strain genotype). Such embodimentscan comprise a single type of hybridization probe which shows differentcomplementarity to different HCV strains, or multiple hybridizationprobes wherein a first probe is substantially complementary with a firstHCV strain genotype and an at least second probe is substantiallycomplementary with a second HCV strain genotype, etc. or multiplehybridization probes that are substantially complementary with multipleareas of those HCV strain genotypes.

Further aspects of the invention comprise kits for identifying one ormore nucleic acid targets in a sample. Such kits can comprise: primersthat are present in unequal amounts that are specific for amplificationof one or more targets; one or more labeled hybridization probes(optionally fluorescently labeled) that are completely or partiallycomplementary to at least one region of the nucleic acid target whereinthe hybridization probes form hybridization complexes with the targetsand which complexes have Tms; one or more labeled 5′-nuclease probes(optionally fluorescently labeled); and instructions for real-timeasymmetric PCR amplification of the targets, for measuring the Tms ofthe hybridization complexes, and for identifying the nucleic acids basedupon the Tms of the hybridization complexes. In some embodiments of suchkits, the 5′-nuclease probe and the hybridization probe are the sameprobe. In some embodiments, the 5′-nuclease probe and the hybridizationprobe are not the same probe, e.g., they can differ in sequence, label,etc.

In other aspects the invention comprises a system, having one or morelabeled hybridization probes (optionally fluorescently labeled); one ormore labeled 5′-nuclease probes (optionally fluorescently labeled); twoor more PCR primers that are present in unequal amounts and which arespecific for amplification of one or more target nucleic acids; one ormore container comprising the probes and primers (as well as kinetic PCRconstituents such as buffers, salts and the like); one or more thermalmodulator that is operably connected to the container and which canmanipulate the temperature in the container; one or more detector thatis configured to detect signals from the hybridization and/or5′-nuclease probes (e.g., fluorescent signals); and, one or morecontroller that is operably connected to the detector and the thermalmodulator and that can comprise one or more instruction sets forcontrolling the thermal modulator and the detector and that can alsocomprise one or more instruction sets for correlating the fluorescentsignals and the temperature in the container with the presence of one ormore target nucleic acid. In some embodiments, the 5′-nuclease probe insaid kits is the same probe as the hybridization probe, while in someembodiments the 5′-nuclease probe and the hybridization probe aredifferent from one another. In some embodiments, the system can furthercomprise a light source effective to excite the fluorescently labeledprobe. In other embodiments, the system can further comprise one or moredevices or subsystems for displaying or processing data obtained by thesystem.

In other aspects, the invention comprises a reaction mixture comprisingkinetic PCR primers present in unequal amounts specific foramplification of at least one nucleic acid target, one or more labeled5′-nuclease probes and one or more labeled hybridization probes whereinthe 5′-nuclease probes and the hybridization probes can be either thesame probe or different probes. In such embodiments, the primers arepresent in different amounts and in some embodiments, the hybridizationprobes are present in a greater amount than the amount of the limitingprimer (i.e., the primer present in the smaller amount).

The invention also provides probes suitable for HCV genotyping, forexample, a nucleic acid comprising a polynucleotide sequence of SEQ IDNO: 3.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not necessarily to be imputed to any related or unrelated case,e.g., to any commonly owned patent or application. Although any methodsand materials similar or equivalent to those described herein can beused in the practice for testing of the present invention, the preferredmaterials and methods are described herein. Accordingly, the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “anoligonucleotide” includes a plurality of oligonucleotides; reference toa “probe” includes mixtures of such probes, and the like.

As used herein, a “sample” refers to any substance containing orpresumed to contain nucleic acid (e.g., from a bacteria, virus, etc.).The sample can be of natural or synthetic origin and can be obtained byany means known to those of skill in the art. Such sample can be anamount of tissue or fluid isolated from an individual or individuals,including, but not limited to, for example, skin, plasma, serum, wholeblood, spinal fluid, saliva, peritoneal fluid, lymphatic fluid, aqueousor vitreous humor, synovial fluid, urine, tears, blood cells, bloodproducts, semen, seminal fluid, vaginal fluids, pulmonary effusion,serosal fluid, organs, bronchio-alveolar lavage, tumors, paraffinembedded tissues, etc. Samples also can include constituents andcomponents of in vitro cell cultures, including, but not limited to,conditioned medium resulting from the growth of cells in the cellculture medium, recombinant cells, cell components, etc. A nucleic acidcan be obtained from a biological sample by procedures well known in theart.

The term “nucleic acid” refers to a polymer of monomers that can becorresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid(DNA) polymer, or analog thereof. This includes polymers of nucleotidessuch as RNA and DNA, as well as modified forms thereof, peptide nucleicacids (PNAs), locked nucleic acids (LNA™s), and the like. In certainapplications, the nucleic acid can be a polymer that includes multiplemonomer types, e.g., both RNA and DNA subunits. A nucleic acid can be orinclude, e.g., a chromosome or chromosomal segment, a vector (e.g., anexpression vector), an expression cassette, a naked DNA or RNA polymer,an amplicon, an oligonucleotide, a primer, a probe, etc. A nucleic acidcan be e.g., single-stranded or double-stranded, or DNA:RNA hybrids, DNAand RNA chimeric structures. Unless otherwise indicated, a particularnucleic acid sequence optionally comprises or encodes complementarysequences, in addition to any sequence explicitly indicated. There is nointended distinction in length between the term “nucleic acid,”“polynucleotide,” and “oligonucleotide,” and the terms can be usedinterchangeably herein unless the context clearly dictates otherwise.Such terms refer only to the primary structure of the molecule.

A nucleic acid is typically single-stranded or double-stranded and willgenerally contain phosphodiester bonds, although in some cases, asoutlined herein, nucleic acid analogs are included that may havealternate backbones, including, for example and without limitation,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925 and thereferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al.(1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) ChemicaScripta 26:1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res.19:1437 and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.(1989) J. Am. Chem. Soc. 111:2321), O-methylphophoroamidite linkages(Eckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press (1992)), and peptide nucleic acid backbones andlinkages (Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992)Chem. Int. Ed. Engl. 31:1008; Nielsen (1993) Nature 365:566; andCarlsson et al. (1996) Nature 380:207), which references are eachincorporated by reference. Other analog nucleic acids include those withpositively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad.Sci. USA 92:6097); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994)Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghvi and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (Jenkins et al. (1995)Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs are alsodescribed in, e.g., Rawls, C & E News Jun. 2, 1997 page 35. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labeling moieties, or toalter the stability and half-life of such molecules in physiologicalenvironments.

In addition to naturally occurring heterocyclic bases that are typicallyfound in nucleic acids (e.g., adenine, guanine, thymine, cytosine, anduracil), nucleic acid analogs also include those having non-naturallyoccurring heterocyclic or other modified bases, many of which aredescribed, or otherwise referred to, herein. In particular, manynon-naturally occurring bases are described further in, e.g., Seela etal. (1991) Helv. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med.Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640.To further illustrate, certain bases used in nucleotides that act asmelting temperature (Tm) modifiers are optionally included. For example,some of these include 7-deazapurines (e.g., 7-deazaguanine,7-deazaadenine, etc.), pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g.,propynyl-dU, propynyl-dC, etc.), and the like. See, e.g., U.S. Pat. No.5,990,303, entitled “SYNTHESIS OF 7-DEAZA-2′-DEOXYGUANOSINENUCLEOTIDES,” which issued Nov. 23, 1999 to Seela. Other representativeheterocyclic bases include, e.g., hypoxanthine, inosine, xanthine; 8-azaderivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine,hypoxanthine, inosine and xanthine; 7-deaza-8-aza derivatives ofadenine, guanine, 2-aminopurine, 2,6-diaminopurine,2-amino-6-chloropurine, hypoxanthine, inosine and xanthine;6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine;5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil, 4-acetylcytosine,5-(carboxyhydroxymethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 7-deazaadenine,2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguanine, 7-deazaguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-Dmannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acidmethylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, and 5-propynyl pyrimidine, and thelike.

Additional examples of modified bases and nucleotides are also describedin, e.g., U.S. Pat. No. 5,484,908, entitled “OLIGONUCLEOTIDES CONTAINING5-PROPYNYL PYRIMIDINES,” issued Jan. 16, 1996 to Froehler et al., U.S.Pat. No. 5,645,985, entitled “ENHANCED TRIPLE-HELIX AND DOUBLE-HELIXFORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDNES,” issued Jul. 8,1997 to Froehler et al., U.S. Pat. No. 5,830,653, entitled “METHODS OFUSING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Nov. 3, 1998 toFroehler et al., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF[2.2.1]BICYCLO NUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al.,U.S. Pat. No. 6,303,315, entitled “ONE STEP SAMPLE PREPARATION ANDDETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct.16, 2001 to Skouv, and U.S. Pat. Application Pub. No. 2003/0092905,entitled “SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al.that published May 15, 2003.

It is not intended that the present invention be limited by the sourceof a nucleic acid, polynucleotide or oligonucleotide. Such nucleic acidcan be from a human or non-human mammal, or any other organism (e.g.,plant, amphibian, bacteria, virus, mycoplasm, etc.), tissue, or cellline, or derived from any recombinant source, synthesized in vitro or bychemical synthesis. Again, the nucleic acid can be DNA, RNA, cDNA,DNA-RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), a hybridor any mixture of the above. The nucleic acid can exist in adouble-stranded, single-stranded or partially double-stranded form. Thenucleic acids of the invention include both nucleic acids and fragmentsthereof, in purified or unpurified forms, including genes, chromosomes,plasmids, the genomes of biological material such as microorganisms,e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals,humans, mycoplasms, and the like.

“Corresponding” as used herein, should be taken to mean identical to, orcomplementary to, a designated sequence of nucleotides in a nucleicacid. The precise meaning of the term will be evident to one of skill inthe art from the context in which it is used.

A “complement” of a nucleic acid (or a nucleic acid that is “specific”in relation to another nucleic acid) refers to at least a nucleic acidsegment that can combine in an antiparallel association or hybridizewith at least a subsequence of that nucleic acid. The antiparallelassociation can be intramolecular, e.g., in the form of a hairpin loopwithin a nucleic acid, or intermolecular, such as when two or moresingle-stranded nucleic acids hybridize with one another. Certain basesnot commonly found in natural nucleic acids may be included in thenucleic acids of the present invention and include, for example,inosine, 7-deazaguanine and those discussed above. Complementarity neednot be perfect (i.e., nucleic acids can be “partially complementary”).Stable duplexes, for example, may contain mismatched base pairs orunmatched bases. Those skilled in the art of nucleic acid technology candetermine duplex stability by empirically considering a number ofvariables including, for example, the length of a region ofcomplementarity, base composition and sequence of nucleotides in aregion of complementarity, ionic strength, and incidence of mismatchedbase pairs.

A “subject” refers to an organism. Typically, the organism is amammalian organism, particularly a human organism. In certainembodiments, for example, a subject is a patient suspected of having agenetic disorder, disease state, or other condition.

Because mononucleotides can be arranged to create oligonucleotides in amanner such that the 5′-phosphate of one mononucleotide pentose ring isattached to the 3′-oxygen of its neighbor in one direction via aphosphodiester linkage, one end of an oligonucleotide is referred to asthe “5′-end” if its 5′-phosphate is not linked to the 3′-oxygen of amononucleotide pentose ring and one end is referred to as the “3′-end”if its 3′-oxygen is not linked to a 5′-phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide, also may be said to have5′ and 3′-ends.

A “primer nucleic acid” or “primer” is a nucleic acid that can hybridizeto a target or template nucleic acid and permit chain extension orelongation using, e.g., a nucleotide incorporating biocatalyst, such asa polymerase under appropriate reaction conditions. Such conditionstypically include the presence of one or more deoxyribonucleosidetriphosphates and the nucleotide incorporating biocatalyst, in asuitable buffer (“buffer” includes substituents which are cofactors, orwhich affect pH, ionic strength, etc.), and at a suitable temperature. Aprimer nucleic acid is typically a natural or synthetic oligonucleotide(e.g., a single-stranded oligodeoxyribonucleotide, etc.). Although otherprimer nucleic acid lengths are optionally utilized, they typicallycomprise hybridizing regions that range from about 6 to about 100nucleotides in length. Short primer nucleic acids generally utilizecooler temperatures to form sufficiently stable hybrid complexes withtemplate nucleic acids. A primer nucleic acid that is at least partiallycomplementary to a subsequence of a template nucleic acid is typicallysufficient to hybridize with the template for extension to occur. Thedesign of suitable primers for, e.g., the amplification of a giventarget sequence is well known in the art and described in the literaturecited herein. A primer nucleic acid can be labeled, if desired, byincorporating a label detectable by, e.g., spectroscopic, photochemical,biochemical, immunochemical, chemical, or other techniques. Toillustrate, useful labels include radioisotopes, fluorescent dyes,electron-dense reagents, enzymes (as commonly used in ELISAs), biotin,or haptens and proteins for which antisera or monoclonal antibodies areavailable. Many of these and other labels are described further hereinand/or otherwise known in the art. One of skill in the art willrecognize that, in certain embodiments, primer nucleic acids can also beused as probe nucleic acids. See, below.

As used herein, the term “probe” refers to an oligonucleotide (or othernucleic acid sequence) which can form a duplex structure with a regionof a target nucleic acid (or amplicon derived from such target nucleicacid), due to partial or complete complementarity of at least onesequence in the probe with a sequence in the target nucleic acid undersuitable conditions. The probe, in certain embodiments, does not containa sequence complementary to sequence(s) of a primer in a 5′-nucleasereaction. As discussed herein, the probe can be labeled or unlabeled.The 3′-terminus of the probe optionally can be “blocked” to prohibitincorporation of the probe into a primer extension product. “Blocking”can be achieved by using non-complementary bases or by adding a chemicalmoiety such as biotin or a phosphate group to the 3′-hydroxyl of thelast nucleotide, which can, depending upon the selected moiety, serve adual purpose by also acting as a label for subsequent detection orcapture of the nucleic acid attached to the label. Blocking can also beachieved by removing the 3′-OH or by using a nucleotide that lacks a3′-OH such as a dideoxynucleotide, or by adding a bulky group thatblocks extension by steric hindrance.

The term “hybridizing region” refers to that region of a nucleic acidthat is exactly or substantially complementary to, and thereforehybridizes to, the target sequence. For use in a hybridization assay,e.g., for the discrimination of single nucleotide differences insequence, the hybridizing region is typically from about 8 to about 100nucleotides in length. Although the hybridizing region generally refersto the entire oligonucleotide, the probe may include additionalnucleotide sequences that function, for example, as linker binding sitesto provide a site for attaching the probe sequence to a solid support orthe like. Probes herein can comprise “5′-nuclease” probes (typicallycomprising a fluorescent label(s) and typically a quencher as well) aFRET probe, or a molecular beacon, or the like, which can also beutilized to detect hybridization between the probe and target nucleicacids in a sample. In some embodiments, the hybridizing region of theprobe is completely complementary to the target sequence. However, ingeneral, complete complementarity is not necessary (i.e., nucleic acidscan be partially complementary to one another); stable duplexes maycontain mismatched bases or unmatched bases. Modification of thestringent conditions may be necessary to permit a stable hybridizationduplex with one or more base pair mismatches or unmatched bases.Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), whichis incorporated by reference, provides guidance for suitablemodification. Stability of the target/probe duplex depends on a numberof variables including length of the oligonucleotide, base compositionand sequence of the oligonucleotide, temperature, and ionic conditions.One of skill in the art will recognize that, in general, the exactcomplement of a given probe is similarly useful as a probe. One of skillin the art will also recognize that, in certain embodiments, probenucleic acids can also be used as primer nucleic acids.

A “5′-nuclease probe” refers to an oligonucleotide that comprises atleast one light emitting labeling moiety and that is used in a5′-nuclease reaction to effect target nucleic acid detection. In someembodiments, for example, a 5′-nuclease probe includes only a singlelight emitting moiety (e.g., a fluorescent dye, etc.). In certainembodiments, 5′-nuclease probes include regions of self-complementaritysuch that the probes are capable of forming hairpin structures underselected conditions. To further illustrate, in some embodiments a5′-nuclease probe comprises at least two labeling moieties and emitsradiation of increased intensity after one of the two labels is cleavedor otherwise separated from the oligonucleotide. In certain embodiments,a 5′-nuclease probe is labeled with two different fluorescent dyes,e.g., a 5′-terminus reporter dye and the 3′-terminus quencher dye ormoiety. In some embodiments, 5′-nuclease probes are labeled at one ormore positions other than, or in addition to, terminal positions. Whenthe probe is intact, energy transfer typically occurs between the twofluorophores such that fluorescent emission from the reporter dye isquenched at least in part. During an extension step of a polymerasechain reaction, for example, a 5′-nuclease probe bound to a templatenucleic acid is cleaved by the 5′ to 3′-nuclease activity of, e.g., aTaq polymerase or another polymerase having this activity such that thefluorescent emission of the reporter dye is no longer quenched.Exemplary 5′-nuclease probes are also described in, e.g., U.S. Pat. No.5,210,015, entitled “Homogeneous assay system using the nucleaseactivity of a nucleic acid polymerase,” issued May 11, 1993 to Gelfandet al., U.S. Pat. No. 5,994,056, entitled “Homogeneous methods fornucleic acid amplification and detection,” issued Nov. 30, 1999 toHiguchi, and U.S. Pat. No. 6,171,785, entitled “Methods and devices forhomogeneous nucleic acid amplification and detector,” issued Jan. 9,2001 to Higuchi, which are each incorporated by reference. In otherembodiments, a 5′-nuclease probe may be labeled with two or moredifferent reporter dyes and a 3′-terminus quencher dye or moiety.

A “hybridization probe” herein refers to a labeled probe used indetermination of Tm. In certain embodiments, the 5′-nuclease probe andthe hybridization probe are the same. In other embodiments, the5′-nuclease probe and the hybridization probe are separate probes andeach can optionally comprise different labels and/or quenchers and eachcan optionally hybridize with different areas of the target nucleicacid. The hybridization probes herein are optionally dual labeled orquenched probes, wherein the quenchers may or may not be fluorescent. Inthe probes, energy transfer occurs between the reporter moiety and thequencher. Such probes can include, e.g., dark quenchers or the like. Insome aspects, the hybridization probe is used as a genotyping probe,where the probe is used to assign a hybridization target to a particulargenotype, e.g., a viral genotype.

A “label” refers to a moiety attached (covalently or non-covalently), orcapable of being attached, to a molecule, which moiety provides or iscapable of providing information about the molecule (e.g., descriptiveor identifying information about the molecule) or another molecule withwhich the labeled molecule interacts (e.g., hybridizes). Exemplarylabels include fluorescent labels (including, e.g., quenchers orabsorbers), non-fluorescent labels, colorimetric labels,chemiluminescent labels, bioluminescent labels, radioactive labels,mass-modifying groups, antibodies, antigens, biotin, haptens, enzymes(including, e.g., peroxidase, phosphatase), and the like. Labels mayprovide signals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like. Labels can be used to provide a detectable (andoptionally quantifiable) signal, and which can be attached to a nucleicacid or protein.

In certain embodiments of the invention, a label is a fluorescent dye orfluorophore. Typically, a particular fluorophore can emit light of aparticular wavelength following absorbance of light of shorterwavelength. The wavelength of the light emitted by a particularfluorophore is characteristic of that fluorophore. Thus, a particularfluorophore can be detected by detecting light of an appropriatewavelength following excitation of the fluorophore with light of shorterwavelength. Fluorescent labels may include dyes that are negativelycharged, such as dyes of the fluorescein family, or dyes that areneutral in charge, such as dyes of the carboxyrhodamine family, or dyesthat are positively charged, such as dyes of the cyanine family or therhodamine family. Other families of dyes that can be used in theinvention include, e.g., polyhalofluorescein-family dyes,hexachlorofluorescein-family dyes, coumarin-family dyes, oxazine-familydyes, thiazine-family dyes, squaraine-family dyes, chelatedlanthanide-family dyes, ALEXA FLUOR® dyes, and BODIPY®-family dyes. Dyesof the fluorescein family include, e.g., FAM, HEX, TET, JOE, NAN andZOE. Dyes of the carboxyrhodamine family include Texas Red, ROX, R110,R6G, and TAMRA. FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, and TAMRAare marketed by Perkin-Elmer (Foster City, Calif.), while Texas Red ismarketed by Molecular Probes, Inc. (Eugene, Oreg.). Dyes of the cyaninefamily include Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 and are marketed byAmersham GE Healthcare (Piscataway, N.J.).

The term “quencher” as used herein refers to a chemical moiety thatabsorbs energy emitted from a fluorescent dye, or otherwise interfereswith the ability of the fluorescent dye to emit light. In oneembodiment, the quencher and fluorescent dye are both linked to a commonpolynucleotide. A quencher can re-emit the energy absorbed from afluorescent dye in a signal characteristic for that quencher and thus aquencher can also be a “label.” This phenomenon is generally known asfluorescent resonance energy transfer or FRET. Alternatively, a quenchercan dissipate the energy absorbed from a fluorescent dye as heat (i.e.,a non-fluorescent quencher). Molecules commonly used in FRET include,for example, fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL,and EDANS. Whether a fluorescent dye is a label or a quencher is definedby its excitation and emission spectra, and the fluorescent dye withwhich it is paired. For example, FAM is most efficiently excited bylight with a wavelength of 494 nm, and emits light with a spectrum of500 to 650 nm, and an emission maximum of 525 nm. FAM is a suitabledonor label for use with, e.g., TAMRA as a quencher that has at itsexcitation maximum at 544 nm. Exemplary non-fluorescent quenchers thatdissipate energy absorbed from a fluorescent dye include the Black HoleQuenchers™ marketed by Biosearch Technologies, Inc. (Novato, Calif.),Eclipse Dark Quenchers from Epoch Biosciences (Bothell, Wash.), and IowaBlack (Integrated DNA Technologies, Coralville, Iowa)

As defined herein, “5′ to 3′ nuclease activity” refers to that activityof a template-specific nucleic acid polymerase including either a 5′ to3′-exonuclease activity (traditionally associated with some DNApolymerases whereby nucleotides are removed from the 5′ end of anoligonucleotide in a sequential manner, e.g., E. coli DNA polymerase Ihas this activity whereas the Klenow fragment does not), or a 5′ to3′-endonuclease activity (wherein cleavage occurs more than onephosphodiester bond (nucleotide) from the 5′-end), or both. Although notintending to be bound by any particular theory of operation, thepreferred substrate for 5′ to 3′-endonuclease activity-dependentcleavage on a probe-template hybridization complex is a displacedsingle-stranded nucleic acid, a fork-like structure. Hydrolysistypically occurs at the phosphodiester bond joining the displaced regionwith the base-paired portion of the strand, as discussed in Holland etal., 1991, Proc. Natl. Acad. Sci. USA 88:7276-80, which is herebyincorporated by reference in its entirety.

As used herein, the term “thermostable” and/or “thermoactive” nucleicacid polymerase refers to an enzyme that is relatively stable and/oractive when heated as compared, for example, to nucleotide polymerasesfrom E. coli, that catalyzes the polymerization of nucleosidetriphosphates. Generally, the enzyme will initiate synthesis at the3′-end of the primer annealed to a target sequence, and will continuesynthesis of a new strand toward the 5′-end of the template, and ifpossessing a 5′ to 3′-nuclease activity, will hydrolyze any intervening,annealed probe, thus, optionally releasing both labeled and unlabeledprobe fragments. Such action will continue until synthesis terminates orprobe fragments melt off the target sequence. A representativethermostable enzyme isolated from Thermus aquaticus (Taq) is describedin U.S. Pat. No. 4,889,818 and a method for using it in conventional PCRis described in Saiki et al., 1988, Science 239:487-91. Those of skillin the art will be familiar with other similar enzymes. Taq DNApolymerase has a DNA synthesis-dependent, strand replacement 5′-3′exonuclease activity. See Gelfand, “Taq DNA Polymerase” in PCRTechnology Principles and Applications for DNA Amplification, Erlich,Ed., Stockton Press, N.Y. (1989), Chapter 2. In solution typically thereis little, if any, degradation of probes by such polymerases.

A “5′-nuclease reaction” or “5′-nuclease assay” of target or template,primer, and probe (e.g., 5′-nuclease probes, etc.) nucleic acids refersto the degradation of a probe hybridized to the template nucleic acidwhen the primer is extended by a nucleotide incorporating biocatalysthaving 5′ to 3′-nuclease activity. 5′-nuclease PCRs, also referred to asTaqMan reactions, are a type of PCR or RT-PCR that utilize a labeledoligonucleotide probe that binds to a single stranded nucleic acidtarget. In 5′-nuclease PCR, the probes are complementary (at least inpart) to one or more regions of the target or targets. The probes canoptionally be labeled with any of a number of moieties, but aretypically labeled with a fluorescent label and a quencher. Thearrangement of the moieties within the probe can also be varied indifferent embodiments. Because the fluorescent moiety is in closeproximity to the quencher on the oligonucleotide probe, its fluorescenceis inhibited.

In addition to the bound probe on the target nucleic acid, 5′-nucleaseassays typically comprise amplification primers to allow amplificationof the region of the target nucleic acid which comprises the boundprobe. The enzyme (typically a DNA polymerase or the like) which has a5′-exonuclease activity, cleaves the labeled probe when it extends thegrowing complementary polymer on the target nucleic acid. Once the probeis cleaved, the label on the probe is no longer in close proximity toits quencher and, thus, a discernable and quantifiable change influorescence can be observed. The observations can be made in real-timeas the amplification proceeds.

Again, of course, many optional variations are possible within the basic5′-nuclease scheme. For example, various permutations and applicationsof 5′-nuclease assays can be used to, e.g., determine copy number,genotype, allelic composition, etc. Further examples of such variationscan be found in, e.g., U.S. Pat. No. 5,210,015 entitled “HomogeneousAssay System Using the Nuclease Activity of a Nucleic Acid Polymerase,”to Gelfand, et al. issued May 11, 1993; U.S. Pat. No. 5,487,972 entitled“Nucleic Acid Detection by the 5′-3′ Exonuclease Activity of PolymerasesActing on Adjacently Hybridized Oligonucleotides,” to Gelfand et al.,issued Jan. 30, 1996; U.S. Pat. No. 5,804,375 entitled “ReactionMixtures for Detection of Target Nucleic Acids,” to Gelfand et al.issued Sep. 8, 1998; and U.S. Pat. No. 6,214,979 entitled “HomogeneousAssay System,” to Gelfand et al., issued Apr. 10, 2001.

One measure of 5′-nuclease (e.g., TaqMan®) assay data is typicallyexpressed as the threshold cycle (C_(t)). Fluorescence levels arerecorded during each PCR cycle and are proportional to the amount ofproduct amplified to that point in the amplification reaction. The PCRcycle when the fluorescence signal is first recorded as statisticallysignificant, or where the fluorescence signal is above some otherarbitrary level (e.g., the arbitrary fluorescence level, or AFL), is thethreshold cycle (C_(t)).

In practice, a C_(t) value is the crossover point between the kineticcurve and an arbitrary threshold of fluorescence. The C_(t) value isinversely proportional to the logarithm of the initial number oftemplate copies. C_(t) values are inversely proportional to the log ofthe initial nucleic acid template concentration and thus may be used tocalculate target copy number.

A “target nucleic acid” refers to a nucleic acid that is amplifiedand/or identified by the current invention (e.g., an amplicon).Typically, a target nucleic acid, or “target,” is one to which a probeand/or a primer(s) binds (e.g., a target optionally comprises one ormore sequence of full complementarity with a primer and/or probe orcomprises a sequence(s) with enough complementarity to one or moreprimer and/or probe to have such primer and/or probe bind to the targetunder appropriate environmental or reaction conditions). Typically, theidentity, genotype, sequence, etc., of the target is to be identified bythe methods of the present invention. A “target primer” and a “targetprobe” refer to a primer and probe, respectively, that can hybridize tothe target nucleic acid.

A “polymorphism” refers to a site or sites of a nucleic acid that cancomprise one of a plurality of genotypes. The polymorphism can be anypolymorphism known to those of skill in the art including possiblemutations, insertions or deletions. The polymorphism can be at one sitewithin the nucleic acid or at multiple sites within the nucleic acid,and may encompass one nucleobase, such as a SNP, or more than onenucleobase. For the purposes of the present invention a “polymorphism”can refer to a polymorphism that is at one site of a nucleic acid or toone particular site of a multiple-site polymorphism. In certainembodiments, the polymorphism need not be well known or even known tothose of skill in the art. The polymorphism can simply be any differencein nucleic acid sequence between a known (e.g., a control) nucleic acidand a target nucleic acid.

To determine “percent complementarity” or “percent identity” of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a firstnucleic acid sequence for optimal alignment with a second nucleic acidsequence). The nucleotides at corresponding nucleotide positions arethen compared. When a position in the first sequence is occupied by acomplementary nucleotide as the corresponding position in the secondsequence, then the molecules are complementary at that position.Likewise, when a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percentcomplementarity (or percent identity) between the two sequences is afunction of the number of complementary positions (or identicalpositions) shared by the sequences divided by the total number ofpositions compared (i.e., percent complementarity=number ofcomplementary overlapping positions/total number of positions of theshorter nucleotide×100; and percent identity=number of identicaloverlapping positions/total number of positions of the shorternucleotide×100).

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul,1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm isincorporated into the NBLAST program of Altschul et al., 1990, J. Mol.Biol. 215:403.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which onehalf of a population of double-stranded polynucleotides or nucleobaseoligomers (e.g., hybridization complexes), in homoduplexes orheteroduplexes (i.e., duplexes that are completely or partiallycomplementary), become dissociated into single strands (under definedionic strength, pH and nucleic acid concentration). The prediction of aT_(m) of a duplex polynucleotide takes into account the base sequence aswell as other factors including structural and sequence characteristicsand nature of the oligomeric linkages. Methods for predicting andexperimentally determining T_(m) are known in the art.

For example, a T_(m) is traditionally determined by a melting curve,wherein a duplex nucleic acid molecule is heated in a controlledtemperature program, and the state of association/dissociation of thetwo single strands in the duplex is monitored and plotted until reachinga temperature where the two strands are completely dissociated. TheT_(m) is read from this melting curve. Alternatively, a T_(m) can bedetermined by an annealing curve, wherein a duplex nucleic acid moleculeis heated to a temperature where the two strands are completelydissociated. The temperature is then lowered in a controlled temperatureprogram, and the state of association/dissociation of the two singlestrands in the duplex is monitored and plotted until reaching atemperature where the two strands are completely annealed. The T_(m) isread from this annealing curve.

The terms “stringent” or “stringent conditions,” as used herein, denotehybridization conditions of low ionic strength and high temperature, asis well known in the art. See, e.g., Sambrook et al., 2001, MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (Ausubel et al., ed., J. Wiley & Sons Inc., New York,1988); Tijssen, 1993, “Overview of principles of hybridization and thestrategy of nucleic acid assays” in Laboratory techniques inbiochemistry and molecular biology: Hybridization with nucleic acidprobes (Elsevier), each of which is hereby incorporated herein byreference. Generally, stringent conditions are selected to be about5-30° C. lower than the thermal melting point (T_(m)) for the specifiedsequence at a defined ionic strength and pH. Alternatively, stringentconditions are selected to be about 5-15° C. lower than the T_(m) forthe specified sequence at a defined ionic strength and pH. For example,stringent hybridization conditions will be those in which the saltconcentration is less than about 1.0 M sodium (or other salts) ion,typically about 0.01 to about 1 M sodium ion concentration at about pH7.0 to about pH 8.3 and the temperature is at least about 25° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 55° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be modified with the addition of hybridization destabilizingagents such as formamide. An exemplary non-stringent or low stringencycondition for a long probe (e.g., greater than 50 nucleotides) wouldcomprise a buffer of 20 mM Tris, pH 8.5, 50 mM KCl, and 2 mM MgCl₂, anda reaction temperature of 25° C.

As used herein, the expression “hepatitis C virus type” refers to thecategorization of a hepatitis C virus (HCV) based on its genomicorganization. The categorization of an HCV isolate into a particulartype category reflects its genomic relatedness to other HCV isolates andits relatively lesser relatedness to other HCV isolates. As used herein,HCV typing nomenclature is consistent with the widely adoptednomenclature proposed by Simmonds et al (1994) Letter, Hepatology19:1321-1324. See, also, Zein (2000) “Clinical Significance of HepatitisC Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235; Maertensand Stuyver (1997) “Genotypes and Genetic Variation of Hepatitis CVirus,” p. 182-233, In Harrison, and Zuckerman (eds.), The MolecularMedicine of Viral Hepatitis, John Wiley & Sons, Ltd., Chichester,England.). The system of Simmonds et al (1994) places the known HCVisolates into one of eleven (11) HCV genotypes, namely genotypes 1through 11. Each genotype is further subdivided into groupings termedsubtypes that reflect relatedness among strains of the same genotype. AnHCV subtype is written by a lowercase roman letter following thegenotype, e.g., subtype 1a, subtype 1c, subtype 6a, etc. Geneticvariants found within an individual isolate are termed quasispecies.Approximately 78 HCV subtypes encompassing all 11 genotypes are knownworldwide; the number of subtypes is not static; as more HCV isolatesare studied and sequenced, it is likely that additional subtypes (andpossibly genotypes) may be recognized.

As used herein, the term “HCV virus type” can refer to either HCVgenotypes or HCV subtypes. As used herein, the term “HCV typing” meansassigning the experimental (e.g., unknown type) HCV to a known genotype(e.g., 1, 2, 3, 4, 5 or 6, or a subset thereof) or assigning theexperimental HCV to a known subtype (e.g., 1a, 1b, 1c, 2a, 2b, 2c, etc.,or a subset thereof).

Some reports (see, e.g., Robertson et al., (1998) Arch. Virol.,143(12):2493-2503) suggest that viral genomic organization is bestrepresented by the creation of viral clades, reflecting the observationthat some HCV genotypes are more closely related to each other than toother HCV genotypes. In this system, clades 1, 2, 4 and 5 correspond togenotypes 1, 2, 4 and 5, while clade 3 comprises genotypes 3 and 10, andclade 6 comprises genotypes 6, 7, 8, 9 and 11. The description of thepresent invention does not use the clade nomenclature.

As used herein, the term “kit” is used in reference to a combination ofarticles that facilitate a process, method, assay, analysis ormanipulation of a sample. Kits can contain written instructionsdescribing how to use the kit (e.g., instructions describing methods forHCV genotyping), chemical reagents or enzymes required for the method,primers and probes, as well as any other components. In someembodiments, the present invention provides kits for HCV typingemploying RT-PCR. These kits can include, for example but not limitedto, reagents for sample collection (e.g., the collection of a bloodsample), reagents for the collection and purification of RNA from blood,a reverse transcriptase, primers suitable for reverse transcription andfirst strand and second strand cDNA synthesis to produce an HCVamplicon, a thermostable DNA-dependent DNA polymerase and freedeoxyribonucleotide triphosphates. In some embodiments, the enzymecomprising reverse transcriptase activity and thermostable DNA-dependentDNA polymerase activity are the same enzyme, e.g., Thermus sp. Z05polymerase or Thermus thermophilus polymerase.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins, eds., 1984); A PracticalGuide to Molecular Cloning (B. Perbal, 1984); and a series, Methods inEnzymology (Academic Press, Inc.), etc. Those of skill in the art willbe familiar with myriad similar references.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a diagram illustrating the placement of primers andprobe upon nucleic acid strands during asymmetric PCR.

FIG. 2 provides a stained agarose gel comparing nucleic acidamplification in asymmetric PCR versus symmetric PCR, using differentprimer ratios with 2×10⁵ copies of cytomegalovirus (CMV) DNA as thetarget nucleic acid.

FIG. 3 provides growth curves generated during an asymmetric kPCR

FIG. 4 provides the first derivative of melting curves using differentprimer ratios in an asymmetric kPCR.

FIG. 5 provides an alignment of a variable region from six different HCVgenotypes and a corresponding exemplary hybridization probe.

FIG. 6 provides growth curve data generated in asymmetric kPCRs of sixdifferent HCV genotypes, using a HEX-labeled probe directed to aconserved region of the HCV genome.

FIG. 7 provides growth curve data generated in asymmetric kPCRs of sixdifferent HCV genotypes, using a FAM-labeled probe directed to avariable region of the HCV genome.

FIG. 8 provides second derivative plot of annealing curves generated inasymmetric kPCRs of six different HCV genotypes using a probe directedto a variable region of the HCV genome.

DETAILED DESCRIPTION

The current invention comprises methods, compositions, kits, systems,and reaction mixtures useful for detection, classification, andquantification of particular nucleic acid sequences within samples(e.g., within clinical samples such as blood or sputum or withinisolated DNA or RNA samples, etc.). The current invention can be usedfor, e.g., classifying, identifying, quantifying, and/or genotypingnucleic acids from various strains of bacteria, viruses, fungi, etc.,especially those involved in infections and diseases (e.g., of humans,livestock, plants, etc.).

The methods of the invention utilize real-time, or kinetic, polymerasechain reaction amplification of target nucleic acid sequences performedin an asymmetric manner in the presence of a labeled oligonucleotideprobe (a 5′-nuclease probe). The asymmetric kinetic PCR results in amixture of single-stranded and double-stranded amplification productshaving an excess of one strand over the other. When the kinetic PCR iscompleted, melting/annealing curves are created using one or morehybridization probes. These melting/annealing curves are used togenerate a Tm for the hybridization probe, which may be used to identifythe target nucleic acid in the sample. In some embodiments, thehybridization probe is present in the kinetic PCR; in other embodiments,the hybridization probe is added after completion of the amplificationreaction. In certain embodiments the 5′-nuclease probes and thehybridization probes (e.g., probes which are used in construction ofthermal curves), comprise different probes (e.g., different in sequenceand/or in binding site, etc.) while in certain other embodiments, the5′-nuclease probes can also act as hybridization probes.

As a result of embodiments wherein the asymmetric kinetic PCR comprisesan amount of the 5′-nuclease probe greater than the amount of thelimiting PCR primer, a portion of a 5′-nuclease probe that hybridizes tothe excess strand is left uncleaved at the finish of the amplificationreaction. This “left-over” probe can then be used to create annealing ormelt curves to determine Tms, which can aid in the determination ofidentity of the target nucleic acid that was amplified.

Incorporating a melting or annealing step into an asymmetric kinetic PCRrequires several considerations. First, in a conventional kinetic PCRthe probe target typically is a double-stranded amplicon (as opposed tojust a single-stranded complement), thus, there are significantcompetitive effects that arise from strand reannealing, especially whenthe amplicon concentration rises in later PCR cycles. Second, since inany kinetic PCR the probe is degraded by the 5′ to 3′-nuclease activityof the DNA polymerase, a supply of such probe must be ensured at the endof PCR in order to generate the melting/annealing curve. Both of thesechallenges can be met in the invention by performing asymmetric kineticPCR. In asymmetric kinetic PCR the concentration of the primer used togenerate the strand to which the 5′-nuclease probe (e.g., the probe usedin the 5′-nuclease reaction) binds is in excess over the concentrationof the other primer. This, thus, produces a greater amount of one strandof the amplicon (or amplified target nucleic acid) than the otherstrand. This, in turn, allows the 5′-nuclease probe to bind to itstarget (on the strand that is in excess) without the other ampliconstrand competing it off. The second, or limiting, primer also limits theamount of probe cleaved during PCR, thus ensuring that there is probeleft behind to perform the post-PCR melting step (in embodiments whereinadditional hybridization probe is not added post-PCR and/or wherein adifferent hybridization probe is used). See FIG. 1. It is surprising howlittle growth curve (i.e., cleaved) signal is lost during the PCR underasymmetric conditions. For only a modest drop in growth curve signal,enough probe can be left behind to give the additional melting data.

5′-Nuclease probes have typically been used to generate fluorescentsignal during real-time or kinetic PCR in the form of a growth curve.From such growth curves a C_(t) (threshold cycle) value is calculatedand used, in either a quantitative or qualitative algorithm, to give adesired result such as a copy number, genotype, or target identity.During this process, the 5′-nuclease probe is cleaved by an enzyme with5′-nuclease activity, thus, generating a variety of DNA fragments, someof which will be labeled with the fluorescent reporter. Once thesefragments are generated they can no longer participate in further signalgeneration. However, by using asymmetric kinetic PCR, full-length intact5′-nuclease probe can be left behind after the PCR is complete, e.g.,when asymmetric PCR is performed, and/or when an excess of probe overlimiting primer is added. Again, by arranging for sufficient probe to beleft after the growth curve is generated, further information can beprovided about the target that has been amplified by performing amelting step or an annealing step. A particular Tm could indicate thatthe probe and the target match perfectly and therefore the targetnucleic acid belongs to a certain virus group or is a certain virus,virus type, or virus subtype, etc. Alternatively, the Tm could be lowerthan that which would result from a perfectly matched probe and target.The degree of decrease in the Tm can indicate (or can allow to becalculated) what mismatches are present, and thus, that the targetsequence belongs to a different virus group.

Tm's generated from the current invention can therefore provideadditional information to that obtained from the growth curve data. Forexample, viral identity in a positive sample in a blood screening assay;bacterial or fungal identity in a microbiology assay; or genotyping,e.g., in an HCV positive sample can all be performed with variousembodiments of the current invention. In certain embodiments, the stepsof the invention are achieved in a single-tube, homogeneous assay,without removing the cap. This greatly adds to the value of bothexisting and future assays requiring only minor changes in primer andprobe concentrations and using existing commercially available5′-nuclease reaction platforms.

During the course of typical 5′-nuclease assays, the probe is cleaved byan enzyme having 5′-nuclease activity (commonly a DNA polymerase such asTaq polymerase, etc.). Cleavage of the probe creates a fluorescentsignal during real-time or kinetic PCR. Such signals are used to createa growth curve (or amplification curve) that can be used to calculate aCT and to determine, e.g., presence or absence, copy number, genotype,target identity, etc. of the nucleic acid from a quantitative orqualitative algorithm. After the probe is cleaved, it typically can nolonger participate in further signal generation. The current inventionprovides a 5′-nuclease assay in which a certain amount of probe is leftover after the 5′-nuclease assay is performed, such that the remainingprobe can be used to generate thermal melting (or thermal annealing)curves because of the change in fluorescence when the labeled probehybridizes or melts from the target nucleic acid. Information about thetarget nucleic acid (e.g., sequence, and thus, genotype, identity, etc.)can be determined from the Tm's generated, separate and apart from theinformation generated by the growth curve. To ensure that sufficientprobe remains, asymmetric PCR is performed with an excess of one primerin comparison to the other.

Asymmetric PCR

The current invention utilizes asymmetric PCR to ensure that sufficientprobe remains after kinetic PCR amplification and that ssDNA is presentfor the probe to bind (either 5′-nuclease probe or a non-5′-nucleasehybridization probe). FIG. 1 schematically illustrates asymmetric PCR asutilized herein. As can be seen in FIG. 1, the probe binds to the excesstarget nucleic acid strand, i.e., the strand that is generated by theexcess primer. PCR occurs after the primers bind, and double strandedtarget is synthesized until the limiting primer is depleted. After thelimiting primer is depleted, linear amplification continues to generatethe excess single strand. Such excess strand (i.e., the strand that isin excess in relation to the other) is the strand to which the probebinds. The strand having the bound probe, i.e., the excess strand, isthe one involved in generating signal from 5′-nuclease release of labelduring the kinetic PCR amplification and is the one involved ingeneration of signal involving hybridization in the post-PCR melt/annealsteps.

A factor in the use of asymmetric PCR concerns the effects of lowconcentration of the limiting primer on generation of the target nucleicacid and on generation of the signal during PCR. To ensure thatasymmetric PCR does not unduly influence the current invention,different primer ratios from 1:1 to 5:1 were evaluated in a CMV5′-nuclease assay. FIG. 2 shows a photograph of a stained gel whichshows that the total amount of target nucleic acid produced was reducedwhen the PCR went from symmetric PCR (here with 30 pmol of each primer)to asymmetric PCR (here 50 pmol of one primer and 10 pmol of the otherprimer). However, the growth curves produced from the asymmetric PCR,shown in FIG. 3, showed very minimal effect on fluorescent signalgeneration from the varying primer amounts. This result demonstratesthat probe cleavage in asymmetric PCR is sufficient to generate a usefulgrowth curve. In certain embodiments, different primer ratios areoptionally used. For example, some embodiments can have primer ratiosranging from at least 2:1, at least 3:1, at least 4:1, at least 5:1, atleast 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, atleast 15:1, at least 20:1, at least 50:1, at least 100:1, or at least200:1 or more.

FIG. 4 shows post-PCR melting curves, e.g., as would be done todetermine identity or genotype of a target, etc. The effect of thedifferent primer ratios on signal generation can be seen from thefigure. Fully symmetric PCR (i.e., wherein the primers are present inequal amounts) gives no melting signal at all, most likely because themajority of the probe has been cleaved during the amplification processor because of competition for amplicon strand re-annealing. AsymmetricPCR below a ratio of 2:1 also gives no signal, likely for similarreasons. With primer ratios from 2:1 to 5:1, differing amounts of meltsignal are observed. The magnitude of signal generated with theasymmetric primers is approximately proportional to the primer ratio. Itwill be appreciated that in certain embodiments, the amount of probeadded is greater than the amount of the limiting primer in theasymmetric PCR. Furthermore, it will be noted that the Tm of the probealso shifts depending on primer ratio. This is most likely due to thedifferent probe concentrations left after PCR since Tm is dependent onoligonucleotide concentration.

Different probe concentrations can be seen by running reactions on laserinduced fluorescent capillary electrophoresis, which detects FAMfluorescence. As the primer ratio increases, more full length uncleavedprobe is left behind after the PCR is complete.

Target Nucleic Acid

As explained above, the target nucleic acid(s) herein can be any nucleicacid of any length whose genotype, identity, sequence, or the like is tobe determined. In certain embodiments, the general identity of thetarget nucleic acid is known but for the genotype of one or morepolymorphisms. For example, a sample may be known to be HCV, but theexact viral type or subtype is unknown. In yet other embodiments, eventhe general identity of the target may not be confidently known. Themethods of the present invention can be used to identify the genotype ofone or more of the polymorphisms. The polymorphisms or particularsequences to be determined can be of any size and at any location in atarget nucleic acid that is between the primer binding sites. Typically,the polymorphisms are not at the ends of the target nucleic acid, buteven those at the ends can be genotyped or determined with the methodsof the current invention.

The target nucleic acid can be obtained from any source known to thoseof skill in the art. For example, it can be obtained from a biologicalsample, e.g., those described above or others. It can be a nucleic acidfrom any natural source, e.g., including a human or a human pathogen orany other natural source. Also, in certain embodiments the targetnucleic acid can be produced by synthetic, semi-synthetic or recombinanttechniques.

In certain embodiments of the invention, the target nucleic acid can beamplified by a number of methods known to those of skill in the art.Such methods are described, for example in Saiki et al., 1988, Science239:487-91, the contents of which are hereby incorporated by referencein their entirety. In certain embodiments, amplification techniques canbe advantageously employed to introduce or alter nucleotide sequences ina target nucleic acid. For instance, if a polymorphism to be identifiedor genotyped is at or near an end of the target nucleic acid, additionalnucleotide sequences can be added to the end of the target nucleic acidto facilitate the methods of the instant invention.

Oligonucleotide Probes

The current invention involves the selection and use of labeled probesto hybridize to specific regions of target nucleic acid. The choice ofsuch probes, e.g., not only their specific sequence, but to which targetarea they should be targeted, depends to a large degree upon thespecific target being assayed. Currently, numerous software programs andother protocols exist to help in choosing and designing 5′-nucleaseprobes or other hybridization probes for various applications. Suchprograms and protocols can optionally be utilized with the currentinvention to choose and design probes for the asymmetric kineticPCR/melting curve assays herein. Of course, visual design and placementof probes is also quite applicable to the current invention as well. Theparameters for design of not only 5′-nuclease probes, but varioushybridization probes to construct thermal melting/annealing curves areextremely familiar to those of skill in the art. Programs which utilizedifferent algorithms and parameter sets and which are useful for suchdesign include, e.g., Visual OMP (DNA Software, Inc., Ann Arbor, Mich.),Oligo 6 (Stratagene, La Jolla, Calif.), Sequencher (Gene Codes, AnnArbor, Mich.), and DNAStar (DNAStar, Inc., Madison, Wis.).

In certain embodiments of the invention, the 5′-nuclease probe and/orthe hybridization probe is labeled with a label that facilitates thedetermination of the identity, genotype, or sequence of a target. Theprobe nucleotide sequence can be of any length sufficient toappropriately hybridize to target region(s) on the target nucleic acidand that can be used to genotype or identify the target nucleic acid.The length of the target probe typically will be chosen to givesufficient thermodynamic stability to ensure hybridization of the probeto its target at the temperature of the annealing step of PCR, etc. Forexample, probes with non-conventional DNA bases may optionally be longeror shorter than those with conventional DNA bases. As another example,probes with A/T-rich sequences will be longer than those with G/C-richsequences, where the Tms are identical. The site of the polymorphism ortarget region can be at any location within the probe nucleotidesequence. In some embodiments, the site of such regions is not at the5′-end of the probe nucleotide sequence.

No matter the individual sequence design of the probes used herein, anumber of different, but not limiting, approaches exist. For example,the same probe may be used for both the 5′-nuclease growth curve and forthe melting/annealing curve. Such a probe is optionally targeted to oneregion of target nucleic acid that is common (at least in somevariation) to all possible samples to be assayed. For example, ingenotyping a virus subtype (e.g., such as illustrated in the Exampleherein) from amongst a number of different possibilities, considerationwill typically be taken in choosing polymorphic areas of the targetnucleic acid that contain sequence motifs unique to each subtype. Thus,the probe will differentially hybridize to such target region dependingon the genotype of the target nucleic acid. Again, such varying degreeof match between the probe and the target in a sample and/or amongst thedifferent genotypes in a sample, influences the Tm curves generated inthe assay and so can allow identification of the nucleic acid sample(s).

Thus, in a hypothetical example, a sample may contain a mixture of anynumber of unidentified nucleic acid types. For example, the sample maycontain possibly related viral strains (e.g., HCV types/subtypes).Probes (5′-nuclease and/or 5′-nuclease and other separate hybridizationprobes) can be designed for a particular polymorphic region on the HCVnucleic acid. Mismatches between the probe(s) and the variouspolymorphic target regions would lead to different Tms (under similarconditions) between the probe(s) and the different target nucleic acids.Based on the sequences of the suspected targets, the expected Tms (underdefined conditions) could be calculated.

The actual Tm curves generated can then be compared against predictedcurves or previously generated standard curves. For example, if virus 1were expected to produce a Tm of X under defined conditions and sample 1did not produce a Tm of X under those conditions, then virus 1 could beruled out as a possible component of the sample mixture.Correspondingly, if the Tm produced from an unknown in a sample produceda Tm of Y, the sequence of the hybridization target area could becalculated based on the known probe sequence, the assay conditions, etc.Determination of the unknown in the sample (e.g., through comparison ofcalculated sequences against sequence databases of known virus, etc.)could then be done.

In certain embodiments herein, the invention can comprise probe(s) usedfor the 5′-nuclease growth curve portion of the analysis that aredifferent from the probe(s) used for the Tm curve generation portion ofthe analysis. Such an arrangement could be useful in situations wherein,e.g., the region of polymorphism on the target nucleic acid is sodiverse that construction of a stable 5′-nuclease probe which would workwithin amplification conditions is not feasible. Thus, for example, aplurality of completely complementary or substantially complementaryprobes or partially complementary probes, each specific for at least oneof the target nucleic acids, could be used for the 5′-nuclease analysis(thus ensuring the measurement of amplification of all desired targets)while a more generalized probe sequence (e.g., one that would hybridizeto a number of related sequences) could be used for generation of Tmcurves from a number of different target sequences. Also, it will beappreciated that while certain embodiments of the invention contain5′-nuclease probes (e.g., in construction of the kinetic PCR growthcurve), in those embodiments wherein different probes are used for thegrowth curve and the melt curve, the probes used in construction of themelt curve need not be 5′-nuclease probes (i.e., such probes are notnecessarily hydrolyzed in certain embodiments). Whether the probes arehydrolyzed or not, can influence their design. For example, some5′-nuclease probes may be about 30 bp in length and contain a dyeinternal to the probe ends. However, in probes that are not hydrolyzedthe length may be shorter, the dye may be located on a terminus, etc.

In certain embodiments of the invention, the 5′-nuclease probe and/orthe hybridization probe is labeled with a label that facilitates thedetermination of the identity, genotype, or sequence of anoligonucleotide fragment. The probe nucleotide sequence can be of anylength sufficient to appropriately hybridize to target region(s) on thetarget nucleic acid and that can be used to genotype or identify thetarget nucleic acid. The length of the target probe typically will bechosen to give sufficient thermodynamic stability to ensurehybridization of the probe to its target at the temperature of theannealing step of PCR, etc. For example, probes with non-conventionalDNA bases may optionally be longer or shorter than those withconventional DNA bases. As another example, probes with A/T-richsequences will be longer than those with G/C-rich sequences. Forexample, since the current invention utilizes 5′-nuclease reactions,which will cleave the 5′-nuclease probes, longer probes can be utilizedthan in non-5′-nuclease reactions. Such longer probes can allow finerdiscrimination between sequences in Tm analysis and a bigger diagnosticwindow. The site of the polymorphism or target region can be at anylocation within the probe nucleotide sequence. In some embodiments, thesite of such regions is not at the 5′-end of the probe nucleotidesequence.

Furthermore, in embodiments wherein different probes are used for the5′-nuclease and Tm curves, the probes are optionally added at the sametime, typically at the start of the analysis. Other embodiments hereincan optionally comprise addition of multiple probes at different timeswithin the analysis. For example, 5′-nuclease curve probe(s) can beadded before the hybridization probe(s) are added.

In some embodiments, the probe nucleotide sequence (whether 5′-nucleaseor hybridization) is identical or complementary to the target region.However, in many embodiments, the probe nucleotide sequence can haveless than 100% identity or complementarity to the target nucleotideregion. In certain embodiments of the invention, the probe nucleotidesequence can have 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% or lesscomplementarity or identity to the target nucleotide region. In certainembodiments of the invention, the probe nucleotide sequence hybridizesto the target nucleotide region under stringent or highly stringentconditions. In other embodiments of the invention, the probe nucleotidesequence hybridizes to the target nucleotide region under low stringencyconditions.

In certain embodiments of the invention, the probe can comprise one ormore label, and optionally one or more quencher. In convenientembodiments, the label can be a label that facilitates the determinationof the kinetic growth curve and/or the Tm curve.

The probe can be labeled by incorporating moieties detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. The method of linking or conjugating the label to theoligonucleotide probe depends, of course, on the type of label(s) and/orquencher(s) used and the position of the such on the probe. Typically,labels provide signals that are detectable by fluorescence, but someembodiments can also comprise labels detectable by, e.g., radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, and the like. See above.

Fluorescent labels can include dyes that are negatively charged, such asdyes of the fluorescein family, or dyes that are neutral in charge, suchas dyes of the rhodamine family, or dyes that are positively charged,such as dyes of the cyanine family. Dyes of the fluorescein familyinclude, e.g., FAM, HEX, TET, JOE, NAN and ZOE. Dyes of the rhodaminefamily include Texas Red, ROX, R110, R6G, and TAMRA. FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, and TAMRA are marketed by Perkin-Elmer (FosterCity, Calif.), and Texas Red is marketed by Molecular Probes, Inc.(Eugene, Oreg.). Dyes of the cyanine family include Cy2, Cy3, Cy5, andCy7 and are marketed by Amersham (Piscataway, N.J.). Other families ofdyes that can be used in the invention include, e.g.,polyhalofluorescein-family dyes, hexachlorofluorescein-family dyes,coumarin-family dyes, oxazine-family dyes, thiazine-family dyes,squaraine-family dyes, chelated lanthanide-family dyes, ALEXA FLUOR®dyes (Molecular Probes, Inc., Eugene Oreg.), and BODIPY®-family dyes(Molecular Probes, Inc.)

In addition to fluorescent label(s), other embodiments may compriseprobes having one or more quencher moieties. A quencher refers to achemical moiety that absorbs energy emitted from a fluorescent dye, orotherwise interferes with the ability of the fluorescent dye to emitlight. A quencher may re-emit the energy absorbed from a fluorescent dyein a signal characteristic for that quencher, thus a quencher can alsobe a label. Alternatively, a quencher may dissipate the energy absorbedfrom a fluorescent dye as heat. Molecules commonly used in FRET include,for example, fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL,and EDANS. Exemplary non-fluorescent quenchers that dissipate energyabsorbed from a fluorescent dye include the Black Hole Quenchers™marketed by Biosearch Technologies, Inc. (Novato, Calif.), Eclipse DarkQuenchers from Epoch Biosciences (Bothell, Wash.), and Iowa Black(Integrated DNA Technologies, Coralville, Iowa).

The labels and/or quenchers can be attached to the oligonucleotide probedirectly or indirectly by a variety of techniques. Depending on theprecise type of label used, the label might be located at the 5′ or3′-end of the probe, or be located internally in the nucleotidesequence. The labels and/or quenchers may be attached directly to anucleotide, or may be attached indirectly via linkers or spacer arms ofvarious sizes and compositions to facilitate signal interactions. Usingcommercially available phosphoramidite reagents, one can produceoligomer probes containing functional groups (e.g., thiols or primaryamines) at either terminus via an appropriately protectedphosphoramidite, and can label them using protocols described in, forexample, PCR Protocols: A Guide to Methods and Applications, ed. byInnis et al., Academic Press, Inc., 1990. It is also possible to attacha detectable moiety at the 3′-terminus of the probe by employing, forexample, polynucleotide terminal transferase to add a desired moiety,such as, for example, cordycepin ³⁵S-dATP, and biotinylated dUTP.

Methods for introducing oligonucleotide functionalizing reagents tointroduce one or more sulfhydryl, amino or hydroxyl moieties into theoligonucleotide probe sequence, typically at the 5′-terminus aredescribed in U.S. Pat. No. 4,914,210. A radioactive (e.g., ³²P-labeled)phosphate group can be introduced at a 5′-terminus of an oligonucleotideby using polynucleotide kinase and [gamma-³²P]ATP. Biotin can be addedto the 5′-end by reacting an aminothymidine residue or alkylaminolinker, introduced during synthesis, with an N-hydroxysuccinimide esterof biotin. Labels at the 3′-terminus of an oligonucleotide may be addedusing polynucleotide terminal transferase to add the desired moiety,such as for example, cordycepin ³⁵S-dATP, and biotinylated dUTP.

Certain nucleotide derivatives may also be used as labels. For example,etheno-dA and etheno-A are known fluorescent adenine nucleotides thatcan be incorporated into an oligonucleotide probe. Similarly, etheno-dCis another analog that could be used in probe synthesis.

In certain embodiments of the invention, a probe is multiply-labeled,with each label individually attached to different locations of theprobe. In another embodiment of the invention, a single probe isdual-labeled with a fluorescent dye (i.e., a label) and a quencher. Whenthe probe is intact, the fluorescence of the label is quenched by thequencher. Cleaving the probe between the label and quencher results inless quenching of the label's emitted fluorescence. An exemplarycombination for this aspect of the invention is the fluorescent dye FAMand the quencher BHQ-2.

HCV Genotyping Probes

The invention provides compositions for HCV genotyping, where thecompositions include at least one HCV genotyping probe that can make anHCV genotype assignment. For example, the invention provides an HCVgenotyping probe, e.g., the HCV genotyping probe HCGT27P5′3′ of SEQ IDNO:3, that is able to differentiate a large number of HCV genotypesbased on Tm discrimination, as illustrated in the Example. This probehas the sequence: EFFGGAALLGFFAGGAFGAFFGGGTCCTJ (SEQ ID NO: 3)

where E=BHQ2; J=cx-FAM; F=propynyl dU; L=propynyl dC.

Although this one sequence is provided, it is not intended that theinvention be limited to this one sequence. It will be apparent to one ofskill in the art that this probe sequence can be readily modified toobtain substantially functionally equivalent probe molecules, i.e.,functionally equivalent molecules that are also able to discriminatemultiple HCV genotypes. For example, the probe provided in SEQ ID NO: 3comprises various labels and/or quenchers (e.g., BHQ2 and cx-FAM). Itwill be immediately apparent to one of skill in the art that thesemoieties can be substituted for other types of labels or label systems,and where the probe will retain its critical property of differentiallybinding a multitude of HCV genotypes and not depart from the essentialfeature of the invention. Indeed, such labels can even be removed, andthe probe still retains its essential property of HCV genotypediscrimination. It is intended that these additional functionallyequivalent variant molecules are within the scope of the presentinvention.

Additional supporting information and detailed description of HCVgenotypes, HCV genotyping probes, methods for using such probes (e.g.,Tm determinations for making HCV genotype assignments), and alternativeembodiments in the design of functionally equivalent probes can all befound in cofiled U.S. Utility patent application Ser. No. ______, filedon Jun. __, 2006, entitled “PROBES AND METHODS FOR HEPATITIS C VIRUSTYPING USING SINGLE PROBE ANALYSIS,” by Gupta and Will, with attorneydocket number 78-001710US; and also in cofiled U.S. Utility patentapplication Ser. No. ______, filed on Jun. __, 2006, entitled “PROBESAND METHODS FOR HEPATITIS C VIRUS TYPING USING MULTIDIMENSIONAL PROBEANALYSIS,” by Gupta and Will, with attorney docket number 78-002910US.The entire content of these two cofiled applications are herebyincorporated by reference in their entirety for all purposes.

T_(m) and Hybridization

One of the benefits of the current invention is that melting/annealingcurves are created from the same reaction sample as the kinetic PCRcurves, e.g., within the same sample container. In various embodimentsherein, the temperature profile of the samples under analysis canoptionally be manipulated in several ways in order to produce themelting/annealing curves. For example, the heating and/or cooling of thesamples in order to construct the melting/annealing curves is typicallydone over a determined range of temperatures. The specific temperaturerange is typically set based upon, e.g., the specific targets/probesunder consideration, their sequences (as much as is known at least), thelength of the probe(s), etc. In addition to the levels of temperaturesin the current invention, the speed of temperature change is alsooptionally variable in different embodiments. The changes in temperatureare optionally continuous, but in some embodiments can be discontinuous.

In interpreting the resulting Tm curve in relation to determining theunderlying target genotype, sequence, etc., reference is optionally madebetween the Tm curve of the target sample(s) and one or more control Tmcurves or between the Tm curve of the samples and the predicted Tm curvethat would be expected given the probe sequence and what is known orassumed to be the target sequence, the reaction conditions, etc. Variousembodiments herein can optionally use one or both of such means tointerpret any Tm curves to determine genotype, sequence, identity, etc.of a target nucleic acid in a sample.

Exemplary Uses of the Invention

The current invention is optionally utilized in diagnostics, e.g., ofmedical conditions. Such conditions can include diagnostics involvinginfectious diseases, as well as noninfectious medical conditions such ascancer, etc. In determination of a causative agent in a disease state ina subject, the current invention can distinguish between causativeagents for disease in situations wherein many diverse agents couldpossibly cause the disease. For example, upper respiratory infections(UR1) are very dangerous for those infected with them and correctdiagnosis of the underlying disease agent is very important for propertreatment. Whether the UR1 is due to a virus, bacteria, etc. greatlyinfluences the proper course of patient treatment. In addition, grampositive and gram negative bacterial infections require differentcourses of treatment. With properly selected primers and probes, thecurrent invention can determine the presence or absence of particularagents within a sample, as well as distinguish between certain agentspresent in a sample.

Other diagnostic uses of the current invention involve differentiationbetween causative agents that are close to one another in sequence. Forexample, as illustrated in the Example herein, HCV subtypes can bedistinguished one from another through use of the current invention.Through use of the proper probes and primers, a host of other relateddisease agents can be differentiated and subjects having such diseaseagents can be treated appropriately based upon such differentiation. Forexample, HIV substrains, HCV strains/substrains, flavivirus types andthe like can all optionally be distinguished by embodiments of thecurrent invention.

In other embodiments, the current invention can optionally be used foridentification of nucleic acids in relation to allele typing for higherorganisms (e.g., as in genetic screening), detection of certain cancers,HLA typing, etc.

Kits, Systems and Reaction Mixtures

In various embodiments, the current invention also comprises kits forthe detection and/or quantification and/or identification of nucleicacids. Such kits can comprise any combination of, e.g., appropriate5′-nuclease and hybridization probes (e.g., based upon the specificnucleic acids to be tested, genotyped, etc.), appropriate primers (e.g.,to amplify the target nucleic acid), reagents and materials for theamplification and identification of the target nucleic acid such asbuffers, nucleotides, salts, etc. The kit optionally further comprisesan instruction set or user manual detailing preferred methods of usingthe kit components for discovery or application of diagnostic sets.Typically, the kit contains, in addition to the above components,additional materials which can include, e.g., instructions forperforming the methods of the invention for detection and/orquantification and/or identification of nucleic acids, packagingmaterial, and one or more containers.

In certain embodiments, the invention also comprises a system for thedetection and/or quantification and/or identification of nucleic acids.Such systems can comprise, e.g., one or more fluorescently labeledhybridization probes; one or more labeled 5′-nuclease probes (which canbe the same as the hybridization probes); two or more kinetic PCRprimers that are specific for amplification of nucleic acid targets andwhich are present in unequal amounts for asymmetric kinetic PCR; one ormore containers for the probes, primers, and other PCR constituents; oneor more thermal modulator that is operably connected (i.e., thermallyconnected) to the container and that can controllably change thetemperature in the container; a detector configured to detect thefluorescent signals from the various probes in the reactions (e.g.,whether hybridization probes, 5′-nuclease probes); a monitor forvisually displaying the data; and a controller that is operablyconnected to the monitor, the detector and/or and the thermal modulatorand which can include instruction sets for controlling the thermalmodulator and the monitor, the detector and/or and the thermalmodulator, as well as instruction sets for correlating the fluorescentsignals and the temperature in the container with the presence of one ormore target nucleic acid. In yet other embodiments wherein the probesare labeled through nonfluorescent means (e.g., radioactive), thedetectors in such systems comprise those that detect such nonfluorescentactivity.

The current invention also comprises one or more reaction mixtureshaving primers specific for amplification of at least one nucleic acidtarget, one or more labeled 5′-nuclease probes and one or more labeledhybridization probes wherein the 5′-nuclease probes and thehybridization probes can be either the same probe or different probes.In such embodiments, the primers are present in different amounts and insome embodiments, the hybridization probes are present in a greateramount than the amount of the limiting primer (i.e., the primer presentin the smaller amount).

Embodiments of the kits, etc., of the invention can include those donein a single tube or container, which allows the reactions to be donewithout opening of the tube/container.

EXAMPLE

The following example is offered to illustrate, but not to limit theclaimed invention. One of skill will recognize a variety of non-criticalparameters that may be altered without departing from the scope of theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the appended claims.

Example Use of the Invention to Determine HCV Genotype

Determining the genotype of an HCV (hepatitis C virus) positive samplehas important clinical significance and utility. Knowing the genotype ofthe specific virus allows physicians to select the correct treatmentregimen. As explained above, the current invention can be used todiscriminate amongst a number of nucleic acid targets (e.g., even onesthat are closely related in sequence) such as HCV genotypes. The methodsof the invention use an oligonucleotide probe that is specificallydesigned to be at least partially complementary to a region of targetsequence divergence (e.g., a region that shows divergence betweendifferent strains, alleles, species, etc.) in conjunction with using thedifferent Tms obtained from post-PCR melting and/or annealing analysisto distinguish the nucleic acid targets.

FIG. 5 shows aligned sequences of 6 different HCV genotypes (namelytypes 1a, 2a, 3a, 4, 5, and 6) along with an exemplary hybridizationprobe capable of use in the current invention to discriminate betweenthe different HCV genotypes.

As indicated previously, the 5′-nuclease probe(s) can optionally use adifferent color-channel than the hybridization probe(s). For example, asseen in FIGS. 6-8, a FAM-labeled hybridization probe (to distinguishbetween different genotypes of HCV) and a HEX-labeled 5′-nuclease probewere utilized with selected RNA transcripts of HCV genotypes 1a, 2a, 3a,4, 5, and 6.

In the current example, the asymmetric PCR sample master mix (i.e., thePCR constituents) consisted of: 2.5% glycerol; 5% DMSO; 50 mM Tricine,pH 8.3; 90 mM potassium acetate; 300 μM dATP, 300 μM dGTP, 300 μM dCTP,550 μM dUTP; 0.1 μM upstream (limiting) primer; 0.5 μM downstream(excess) primer; 0.2 μM hydrolysis probe, 0.2 μM hybridization probe; 10U uracil-N-glycosylase; 40 U ZO5 DNA polymerase; and 2.7 mM manganeseacetate.

Template RNA for generating HCV amplicons by RT-PCR was derived by invitro transcription from plasmids carrying HCV genomic material insertscorresponding to types 1a, 2a, 3a, 4, 5 and 6. The sequences of theseinserts correspond to the consensus sequences of each of the respectivetypes as described in FIG. 5. Following the in vitro transcription, theRNA was purified by oligo-dT-sepharose chromatography.

In the example, the hybridization/genotyping probe was present at twicethe concentration of the limiting primer to ensure that not all theprobe was cleaved. The excess primer was present at 5× the limitingprimer concentration to ensure an excess of single-stranded amplicon forthe hybridization probe to bind to. The thermocycling profile used forthe example was: 50° C. for 5 minutes (UNG step); 59° C. for 30 minutes(RT step); 94° C. for 20 seconds−58° C. for 40 seconds×60 cycles; 94° C.for 60 seconds; and 40° C. to 90° C. in 1° C. steps—melt step. Theoligonucleotides included in the reaction were as follows: SEQ ID NameSequence NO: upstream GCAGAAAGCGTCTAGCCATGGCGTTE 1 primer where E =t-butyl benzyl dA downstream GCAAGCACCCTATCAGGCAGTACCACAE 2 primer (thewhere E = t-butyl benzyl dA RT primer) HCGT27P5′3′EFFGGAALLGFFAGGAFGAFFGGGTCCTJ 3 hybridiza- where E = BHQ2; J = cx-FAM;tion/geno- F = propynyl dU; L = propynyl dC typing probe hydrolysisECTCACCGGTJCCGCAGACCACTATGGCTCTCCCP 4 probe (i.e., where E = CY5; J =HEX; 5′-nuclease P = phosphate probe)

The growth curve data from the HEX-labeled 5′-nuclease probe is shown inFIG. 6. In certain embodiments, such probe is a conserved probe whichhas no mismatches between the probe and the selected nucleic acidtranscripts used and should therefore detect all genotypes equally. Thegrowth curve data from the FAM-labeled hybridization probe, however, asshown in FIG. 7, arise from the probe which does not show completecomplementarity to all transcripts tested and therefore shows much morevariability. The reaction with subtypes 2a and 3a, which have thegreatest amount of mismatch between the probe and the transcripts,generate very little 5′-nuclease signal.

FIG. 8 shows post-PCR annealing data for each transcript and thehybridization/genotyping probe. In the current example, themelting/annealing curves were produced on an ABI Prism 7700Thermocycler. Those of skill in the art will be quite familiar withbasic protocols and associated parameters (e.g., control of stringency,thermocycler, etc.) for construction of thermal melting/annealing curvesbetween nucleic acids. As can be seen, a wide range of Tms exist betweenthe different HCV genotypes, e.g., between 50° C. and 78° C. Asexplained previously, the higher Tms arise from matches between thehybridization probe and transcripts from HCV genotypes that are closelymatched in sequence, while those genotypes that have less sequence matchwith the probe in such region show lower Tm. The range of different Tmsseen in FIG. 8 allows for easy differentiation between the differentgenotypes. While the Tms for genotype 2a and genotype 3a are closer toeach other than the other readings, selection of a different genotypingprobe (e.g., different in sequence, but binding to the same region orbinding to a different region on the transcripts) could optionally beused to further clarify the difference between the genotypes in thesample. Also, changes in reaction conditions (e.g., temperature, saltconcentrations, etc.) could also optionally be used to helpdifferentiate such. FIG. 8 illustrates that, as with allmelting/annealing based assays, performance is greatly affected by newor unknown mismatches in the target region. Different embodiments hereincan optionally account for such new or unknown mismatches in probebinding regions by constructing a variety of hybridization probes, etc.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1. A method for identifying one or more nucleic acid targets in asample, the method comprising: a) performing an asymmetric kinetic PCRin a reaction mixture containing one or more labeled 5′-nuclease probesand one or more labeled hybridization probes wherein the 5′-nucleaseprobes and the hybridization probes can be the same probe or differentprobes; b) monitoring one or more growth curves from the kinetic PCR; c)modifying the temperature of the reaction mixture after the kinetic PCRto cause a change in association between the one or more labeledhybridization probes and the one or more nucleic acid targets; d)monitoring one or more fluorescent signals generated from the one ormore labeled hybridization probes thereby producing a melting curve orannealing curve; e) correlating the melting curve or annealing curve ofstep d) to a melting or annealing curve of a completely complementaryprobe of one or more known nucleic acid targets, thus, identifying theone or more nucleic acid targets in the sample.
 2. The method of claim1, wherein identifying the one or more nucleic acid targets comprisesidentifying one or more organisms or organism strains having saidnucleic acids.
 3. The method of claim 1, wherein the asymmetric kineticPCR comprises a first primer and at least a second primer, wherein theamount of the first primer is greater than the amount of the secondprimer and wherein the hybridization probe is present in an equal orgreater amount than the second primer.
 4. The method of claim 1, whereinthe asymmetric kinetic PCR comprises a first primer and at least asecond primer, and wherein the PCR comprises at least a 2:1 ratio offirst primer to second primer.
 5. The method of claim 1, wherein theasymmetric kinetic PCR comprises a first primer and at least a secondprimer, and wherein the PCR comprises at least a 3:1 ratio of firstprimer to second primer.
 6. The method of claim 1, wherein theasymmetric kinetic PCR comprises a first primer and at least a secondprimer, and wherein the PCR comprises at least a 4:1 ratio of firstprimer to second primer.
 7. The method of claim 1, wherein theasymmetric kinetic PCR comprises a first primer and at least a secondprimer, and wherein the PCR comprises at least a 5:1 ratio of firstprimer to second primer.
 8. The method of claim 1, wherein the reactionmixture comprises one or more fluorescently labeled 5′-nuclease probes.9. The method of claim 1, wherein the sequence of the one or morelabeled 5′-nuclease probes is the same as the sequence of the one ormore labeled hybridization probes.
 10. The method of claim 1 wherein theone or more labeled 5′-nuclease probes are different from the one ormore labeled hybridization probes.
 11. The method of claim 1, wherein atleast one of the one or more hybridization probes is completelycomplementary to at least one region of at least one nucleic acidtarget.
 12. The method of claim 1, wherein at least one of the one ormore hybridization probes is partially complementary to at least oneregion of at least one nucleic acid target.
 13. The method of claim 1,wherein the one or more hybridization probes are present during thekinetic PCR.
 14. The method of claim 1, wherein the one or morehybridization probes are not present during the kinetic PCR.
 15. Themethod of claim 1, wherein monitoring one or more fluorescent signalscomprises monitoring over a range of temperatures.
 16. The method ofclaim 1, wherein the asymmetric kinetic PCR is monitored by a firstfluorescence and wherein monitoring of the change in association of thehybridization probes is monitored by a second fluorescence which secondfluorescence is different from said first fluorescence.
 17. The methodof claim 1, wherein the asymmetric kinetic PCR is monitored by a firstfluorescence and wherein monitoring of the change in association of thehybridization probes is monitored by a second fluorescence which secondfluorescence is the same as said first fluorescence.
 18. The method ofclaim 1, wherein the one or more nucleic acid targets comprise hepatitisC virus (HCV) nucleic acid.
 19. The method of claim 18, whereinidentifying the one or more HCV nucleic acid target identifies one ormore HCV strain in the sample.
 20. The method of claim 1, wherein atleast one of the hybridization probes is substantially complementary toan HCV strain genotype.
 21. The method of claim 1, wherein the reactionmixture comprises a first hybridization probe and at least a secondhybridization probe, wherein the first hybridization probe issubstantially complementary to a first HCV strain genotype and the atleast a second hybridization probe is substantially complementary to asecond HCV strain genotype.
 22. A kit for identifying one or morenucleic acid targets in a sample, the kit comprising: at least one pairof kinetic PCR primers present in unequal amounts specific foramplification of at least one nucleic acid target; one or morefluorescently labeled hybridization probes at least partiallycomplementary to at least one region of at least one nucleic acidtarget, and instructions for performing a real-time asymmetric PCRamplification of the targets.
 23. A system, comprising: a) one or morefluorescently labeled hybridization probes; b) two or more kinetic PCRprimers present in unequal amounts, said primers being effective toamplify one or more target nucleic acids; c) one or more containercomprising said probes and primers; d) one or more thermal modulatoroperably connected to the container, which modulator manipulatestemperature in the container; e) one or more light sources effective toexcite the labels of the fluorescently labeled hybridization probes; f)one or more detector configured to detect one or more fluorescentsignals from said hybridization probes; and, g) one or more controlleroperably connected to the detector and the thermal modulator, whichcontroller comprises one or more instruction sets for controlling thethermal modulator and the detector and which controller comprises one ormore instruction sets for correlating the one or more fluorescentsignals and the temperature in the container with the presence of one ormore target nucleic acid.
 24. The system of claim 23, further comprisinga labeled 5′-nuclease probe, wherein said 5′-nuclease probe is not thesame sequence as said one or more hybridization probes.
 25. A reactionmixture, comprising two or more kinetic PCR primers present in unequalamounts specific for amplification of at least one nucleic acid target;one or more labeled 5′-nuclease probes and one or more labeledhybridization probes.
 26. A nucleic acid comprising a polynucleotidesequence of SEQ ID NO: 3.